[Show abstract][Hide abstract] ABSTRACT: The mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q --> TAA, 242R --> TGA, 317L --> TAG). The nonsense mutations in SUP45 gene were shown to be situated in moderate contexts for readthrough efficiency. We showed that readthrough efficiency of some of the mutations present in the sup45 mutants is not correlated with full-length Sup45 protein amount. This resulted from modification of both sup45 mRNA stability which varies 3-fold among sup45-n mutants and degradation rate of mutant Sup45 proteins. Our results demonstrate that some substitutions in the place of PTCs decrease Sup45 stability. The viability of sup45 nonsense mutants is therefore supported by diverse mechanisms that control the final amount of functional Sup45 in cells.
[Show abstract][Hide abstract] ABSTRACT: The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3.
In this work we have used previously isolated sup45 mutants of S. cerevisiae to characterize degradation of aberrant mRNA in conditions when translation termination is impaired. We have sequenced his7-1, lys9-A21 and trp1-289 alleles which are frequently used for analysis of nonsense suppression. We have established that sup45 nonsense and missense mutations lead to accumulation of his7-1 mRNA and CYH2 pre-mRNA. Remarkably, deletion of the UPF1 gene suppresses some sup45 phenotypes. In particular, sup45-n upf1Delta double mutants were less temperature sensitive, and more resistant to paromomycin than sup45 single mutants. In addition, deletion of either UPF2 or UPF3 restored viability of sup45-n double mutants.
This is the first demonstration that sup45 mutations do not only change translation fidelity but also acts by causing a change in mRNA stability.
[Show abstract][Hide abstract] ABSTRACT: Abstract
Termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs) – eRF1 and eRF3. The highly conserved translation termination factor eRF1 in Saccharomyces cerevisiae is encoded by the essential gene SUP45 .
We have isolated five sup45-n (n from nonsense) mutations that cause nonsense substitutions in the following amino acid positions of eRF1: Y53 → UAA, E266 → UAA, L283 → UAA, L317 → UGA, E385 → UAA. We found that full-length eRF1 protein is present in all mutants, although in decreased amounts. All mutations are situated in a weak termination context. All these sup45-n mutations are viable in different genetic backgrounds, however their viability increases after growth in the absence of wild-type allele. Any of sup45-n mutations result in temperature sensitivity (37°C). Most of the sup45-n mutations lead to decreased spore viability and spores bearing sup45-n mutations are characterized by limited budding after germination leading to formation of microcolonies of 4–20 cells.
Nonsense mutations in the essential gene SUP45 can be isolated in the absence of tRNA nonsense suppressors.
[Show abstract][Hide abstract] ABSTRACT: The termination of protein synthesis in eukaryotes involves at least two polypeptide release factors (eRFs), eRF1 and eRF3. In mammals two genes encoding eRF3 structural homologues were identified and named GSPT1 and GSPT2.
In the present study, we demonstrate that mouse mGSPT2 but not mGSPT1 could functionally substitute the essential yeast gene SUP35. However, we show that the complementation property of mGSPT1 protein is modified when NH2-tagged by GST. Since mGSPT1 and mGSPT2 differ mainly in their N-terminal regions, we developed a series of N-terminal deleted constructs and tested them for complementation in yeast. We found that at least amino acids spanning 84-120 of mGSPT1 prevent the complementation of sup35 mutation. The fact that chimeras between mGSPT1, mGSPT2 and yeast Sup35 complement the disruption of the SUP35 gene indicates that the N-terminal region of mGSPT1 is not sufficient by itself to prevent complementation. Complementation of the mutant with a double disruption of SUP35 and SUP45 genes is obtained when mGSPT2 and human eRF1 are co-expressed but not by co-expression of mGSPT1 and human eRF1.
Our results strongly suggest that the two proteins (mGSPT1 and mGSPT2) are different. We hypothesize that the full length mGSPT1 does not have the properties expected for eRF3.
Genes to Cells 11/2002; 7(10):1043-57. · 2.73 Impact Factor